J Doog (jd at mesher.cam.ac.uk) wrote:
: Dear Reader
: I am trying to study the expression of a protein whose mass is
: approximately 290 kDa. As it is so large, visualisation on an
: SDS-polyacrylamide gel followed by Western blotting has proved difficult.
: The highest marker in the range is 'only' 200kD, which means that I am
: unable to confirm the size of bands which I see. Bands which may indicate
: that expression is occuring remain in the stacking gel. Have you ever
: tried to do something similar, and if so do you have any advice?
: Thank you in advance
: James Good
: e-mail : jsg at mole.bio.cam.ac.uk
James,
I spent 5 years of grad school working with fibronectin and laminin, and
maybe 7 years working on spectrin. I agree with Ellen on the 5% gel
idea--it served me well in my days with extracellular matrix. To resolve
alpha/beta spectrin (260--280/220--240 Kd) we used an 8% low bis gel and
it did a heck of a job. I believe Dubreuil et al, 1987. J. Cell Biol.
pp 2095--2102 spells it out. If not, E-mail me and I'll send or post the
info. I run the 8x10 mini-gels and blot them in a Hoefer apparatus,
100 volts for two hours. My transfer buffer is like Towbin, but ethanol
is substituted for methanol and the SDS concentration is 0.1%.
If you have material hanging up in the stacker you may try having a
reducing agent present in every step of your lysate/isolation. Also, try
heating your sample to 80 ' C. or so for 10 minutes rather than boiling.
It works for erythrocyte proteins in minimizing that crud.
Regards,
Steve Dahl
